In the past project period cDNAs were cloned for two additional sialyltransferases, the Galbeta1,3GalNAc alpha2,3 sialyltransferase and the Galbeta1,3/4GlcNAc alpha2,3 sialyltransferase. Comparison of the coding sequences with that of the Galbeta1,4GlcNAc alpha2,6 sialyltransferase revealed a unique pattern of homology among glycosyltransferase families. There was a 55 residue stretch of high homology in the center of each enzyme (40-60% identity) with no detectable homology between any of the enzymes outside that region. We have termed this conserved region the sialylmotif. In the next project period several aims pertain to this conserved motif. 1) Additional members of the 10-12 member sialyltransferase family will be cloned using a PCR homology approach using the information contained in the sialylmotif. Preliminary efforts using this approach have been successful in pulling out another cDNA from embryonic brain that has the sialylmotif and other sequence features like the three sialyltransferases cloned to date. A 'soluble form' of this putative enzyme will be constructed and expressed to evaluate the activity and specificity in order to identify which enzyme has been cloned. Other sialyltransferase cDNAs obtained by this approach will be evaluated similarly. 2) The sialylmotif will be investigated for its functional properties by a site- directed mutagenesis expression approach. The driving force for this investigation stems from the successful crystallization of the Galbeta1,4GlcNAc alpha2,6 sialyltransferase by the group of Dr. Don Wiley at Harvard. Thus, the results regarding function will likely be interpretable from the three dimensional structure of the enzyme in the foreseeable future. Several other specific aims take advantage of the new tools resulting from the cloned sialyltransferases. 1) The long time collaboration with Dr. Jurgen Roth for localization of sialyltransferases will be extended to the newly cloned enzymes. 2) The newly expressed enzymes will be used to develop a flexible and practical route to the combined chemical and enzymatic synthesis of sialosides. 3) One of the three cloned sialyltransferases appears to be responsible for the conversion of peanut agglutinin positive (PNA+) T-cells to the PNA- phenotype during maturation in the thymus. In collaboration with Dr. Steve Hedrick and Dr. Ajit Varki at UCSD, transgenic mice are being constructed to test the function of this glycosylation change.